Inhibition of Plasmepsin V Activity Demonstrates Its Essential Role in Protein Export,PfEMP1 Display,and Survival of Malaria Parasites

The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of P. falciparum–infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target.     

Epigenetic silencing of Na,K-ATPase β1 subunit gene ATP1B1 by methylation in clear cell renal cell carcinoma

The Na,K-ATPase or sodium pump carries out the coupled extrusion of Na⁺ and uptake of K⁺ across the plasma membranes of cells of most higher eukaryotes. We have shown earlier that Na,K-ATPase-β₁ (NaK-β) protein levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. The mechanism(s) regulating the expression of NaK-β in tumor tissues has yet to be explored. We hypothesized that DNA methylation plays a role in silencing the NaK-β gene (ATP1B1) expression in kidney cancers. In this study, to the best of our knowledge we provide the first evidence that ATP1B1 is epigenetically silenced by promoter methylation in both renal cell carcinoma (RCC) patients’ tissues and cell lines. We also show that knockdown of the von Hippel-Lindau (VHL) tumor suppressor gene in RCC cell lines results in enhanced ATP1B1 promoter AT hypermethylation, which is accompanied by reduced expression of NaK-β. Furthermore, treatment with 5-Aza-2′-deoxycytidine rescued the expression of ATP1B1 mRNA as well as NaK-β protein in these cells. These data demonstrate that promoter hypermethylation is associated with reduced NaK-β expression, which might contribute to RCC initiation and/or disease progression.     

Linkage mapping in the oilseed crop Jatropha curcas L. reveals a locus controlling the biosynthesis of phorbol esters which cause seed toxicity

Current efforts to grow the tropical oilseed crop Jatropha curcas L. economically are hampered by the lack of cultivars and the presence of toxic phorbol esters (PE) within the seeds of most provenances. These PE restrict the conversion of seed cake into animal feed, although naturally occurring ‘nontoxic’ provenances exist which produce seed lacking PE. As an important step towards the development of genetically improved varieties of J. curcas, we constructed a linkage map from four F₂ mapping populations. The consensus linkage map contains 502 codominant markers, distributed over 11 linkage groups, with a mean marker density of 1.8 cM per unique locus. Analysis of the inheritance of PE biosynthesis indicated that this is a maternally controlled dominant monogenic trait. This maternal control is due to biosynthesis of the PE occurring only within maternal tissues. The trait segregated 3 : 1 within seeds collected from F₂ plants, and QTL analysis revealed that a locus on linkage group 8 was responsible for phorbol ester biosynthesis. By taking advantage of the draft genome assemblies of J. curcas and Ricinus communis (castor), a comparative mapping approach was used to develop additional markers to fine map this mutation within 2.3 cM. The linkage map provides a framework for the dissection of agronomic traits in J. curcas, and the development of improved varieties by marker‐assisted breeding. The identification of the locus responsible for PE biosynthesis means that it is now possible to rapidly breed new nontoxic varieties.     

Effects of single nucleotide polymorphisms on human N-acetyltransferase 2 structure and dynamics by molecular dynamics simulation

Background

Arylamine N-acetyltransferase 2 (NAT2) is an important catalytic enzyme that metabolizes the carcinogenic arylamines, hydrazine drugs and chemicals. This enzyme is highly polymorphic in different human populations. Several polymorphisms of NAT2, including the single amino acid substitutions R64Q, I114T, D122N, L137F, Q145P, R197Q, and G286E, are classified as slow acetylators, whereas the wild-type NAT2 is classified as a fast acetylator. The slow acetylators are often associated with drug toxicity and efficacy as well as cancer susceptibility. The biological functions of these 7 mutations have previously been characterized, but the structural basis behind the reduced catalytic activity and reduced protein level is not clear.

Methodology/Principal Findings

We performed multiple molecular dynamics simulations of these mutants as well as NAT2 to investigate the structural and dynamical effects throughout the protein structure, specifically the catalytic triad, cofactor binding site, and the substrate binding pocket. None of these mutations induced unfolding; instead, their effects were confined to the inter-domain, domain 3 and 17-residue insert region, where the flexibility was significantly reduced relative to the wild-type. Structural effects of these mutations propagate through space and cause a change in catalytic triad conformation, cofactor binding site, substrate binding pocket size/shape and electrostatic potential.

Conclusions/Significance

Our results showed that the dynamical properties of all the mutant structures, especially in inter-domain, domain 3 and 17-residue insert region were affected in the same manner. Similarly, the electrostatic potential of all the mutants were altered and also the functionally important regions such as catalytic triad, cofactor binding site, and substrate binding pocket adopted different orientation and/or conformation relative to the wild-type that may affect the functions of the mutants. Overall, our study may provide the structural basis for reduced catalytic activity and protein level, as was experimentally observed for these polymorphisms.     

Human gingival derived neuronal cells in the optimized caffeic acid hydrogel for hemitransection spinal cord injury model

Spinal cord injury induces scar formation causes axonal damage that leads to the degeneration of axonal function. Still, there is no robust conceptual design to regenerate the damaged axon after spinal injury. Therefore, the present study demonstrates that human gingival derived neuronal stem cells (GNSCs) transplants in the injectable caffeic acid bioconjugated hydrogel (CBGH) helps to bridge the cavity and promote the engraftment and repopulation of transplants in the injured spinal tissue. Our study reports that the bioluminescence imaging in vivo imaging system (IVIS) provides a satisfactory progression in CBGH-GNSCs transplants compare to lesion control and CBGH alone. Immune regulators interleukin-6 (IL-6), tumor necrosis factor-α, neutrophil elastase are decreased, IL-10 is increased. Likewise, immunostaining (TAU/TUJ-1, SOX-2/NeuN, MAP-2/PSD93, NSE, S100b, and GFAP) shown repopulated cells. Also, TRA-1-81 expression confirms the absence of immune rejection in the CBGH-GNSCs transplants. However, locomotor recovery test, gene (IL-6, CASPASE3, p14-ARF, VEGF, LCAM, BDNF, NT3, NGN2, TrKc, FGF2, Sox-2, TUJ-1, MAP-2, Nestin, and NeuN) and protein expression (TAU, TUJ-1, SOX-2 MAP-2, PSD93, NeuN, TRA-1-81, GFAP, TAU, and MBP) shows functional improvements in the CBGH-GNSCs group. Further, GABA and glutamine level demonstrates the new synaptic vesicle formation. Hence, the CBGH scaffold enhances GNSCs transplants to restore the injured spinal tissue.     

Neuroprotective potential of ethanolic extract of Pseudarthria viscida (L) Wight and Arn against beta-amyloid(25-35)-induced amnesia in mice

The neuroprotective potential of ethanolic extract of roots of Pseudarthria viscida (L) Wight and Arn (EEPV) was investigated against beta-amyloid(25-35)-induced amnesia in mice which is a suitable animal model for Alzheimer's disease (AD). The senile plaques of beta-amyloid (Abeta) are major constituents accumulated during the progression of AD as a potent neurotoxicant. In our investigation, intracerebroventricular injection of Abeta(25-35) in mice induced the neurodegeneration, exhibited the increased time of escape latency in behavioral pattern using water maze and decreased the levels of antioxidants namley superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and vitamin C with elevated level of acetylcholinesterase enzyme (AChE). The neuroprotective potential of EEPV was determined by behavioral pattern using water maze and biochemical parameters such as SOD, CAT and GPx and vitamin C content as well as AChE. Mice were treated with EEPV at 200 and 400 mg/kg doses for 21 days. Except control, all animals received a single injection of neurotoxicant Abeta(25-35) on 14th day. In behavioural assessment, treatment with ethanolic extract improved the cognitive function in the water maze and attenuated the elevated levels of AChE with increase in antioxidant enzymes, indicating the neuroprotection with increased levels of vitamin C. These findings suggest that ethanolic extract of P. viscida exerts anti-amnesiac effects and enhances cognitive function.     

Photoperiod influences endogenous indoleamines in cultured green alga Dunaliella bardawil

Effect of light intensity and photoperiod on growth, indoleamines and carotenoid production was studied in unicellular green algae D. bardawil. Maximum biomass and carotenoid contents were found when cultures were grown in light (intensity of 2.0 Klux) at a photoperiod of 16/8h light and dark cycle. There was a profound influence of tested photoperiod conditions of light:dark viz. 8:16, 10:14, and 12:12 hr, continuous light on indoleamines (SER and MEL) production as estimated by HPLC and confirmed by mass spectral data obtained from LC-MS-ESI studies. Serotonin level increased from 908 to 1765 pg/g fresh wt with increase in light duration and melatonin level increased from 267 to 584 pg/g fresh wt during increase in dark phase. Carotenoids production was high in continuous light than other tested conditions.     

Human alpha B-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in mice

The autosomal dominant mutation in the human alphaB-crystallin gene inducing a R120G amino acid exchange causes a multisystem, protein aggregation disease including cardiomyopathy. The pathogenesis of cardiomyopathy in this mutant (hR120GCryAB) is poorly understood. Here, we show that transgenic mice overexpressing cardiac-specific hR120GCryAB recapitulate the cardiomyopathy in humans and find that the mice are under reductive stress. The myopathic hearts show an increased recycling of oxidized glutathione (GSSG) to reduced glutathione (GSH), which is due to the augmented expression and enzymatic activities of glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase, and glutathione peroxidase. The intercross of hR120GCryAB cardiomyopathic animals with mice with reduced G6PD levels rescues the progeny from cardiac hypertrophy and protein aggregation. These findings demonstrate that dysregulation of G6PD activity is necessary and sufficient for maladaptive reductive stress and suggest a novel therapeutic target for abrogating R120GCryAB cardiomyopathy and heart failure in humans.     

Structural analysis and prediction of protein mutant stability using distance and torsion potentials: role of secondary structure and solvent accessibility

Platelet-activating factor receptor (PAFR) is a member of G-protein coupled receptor (GPCR) superfamily. Understanding the regulation mechanisms of PAFR by its agonists and antagonists at the atomic level is essential for designing PAFR antagonists as drug candidates for treating PAF-mediated diseases. In this study, a 3D model of PAFR was constructed by a hierarchical approach integrating homology modeling, molecular docking and molecular dynamics (MD) simulations. Based on the 3D model, regulation mechanisms of PAFR by agonists and antagonists were investigated via three 8-ns MD simulations on the systems of apo-PAFR, PAFR-PAF and PAFR-GB. The simulations revealed that binding of PAF to PAFR triggers the straightening process of the kinked helix VI, leading to its activated state. In contrast, binding of GB to PAFR locks PAFR in its inactive state.